Poor Regenerative Outcome after Skeletal Muscle Necrosis Induced by Bothrops asper Venom: Alterations in Microvasculature and Nerves

et al. (2011) Poor Regenerative Outcome after Skeletal Muscle Necrosis Induced by Bothrops asper Venom: Alterations in Microvasculature and Nerves. PLoS ONE 6(5): e19834. doi:10.1371/journal.pone.0019834 Poor Regenerative Outcome after Skeletal Muscle Necrosis Induced by Bothrops asper Venom: Alterations in Microvasculature and Nerves Rosario Herna ndez 0 Carmen Cabalceta 0 Patricia Saravia-Otten 0 Alessandra Chaves 0 Jose Mara Gutie rrez 0 Alexandra Rucavado 0 Amy C.Y. Lo, The University of Hong Kong, Hong Kong 0 1 Facultad de Ciencias Qu micas y Farmacia, Universidad de San Carlos de Guatemala, Finca San Isidro, Guatemala, 2 Instituto Clodomiro Picado, Facultad de Microbiolog a, Universidad de Costa Rica , San Jose , Costa Rica Background: Viperid snakebite envenoming is characterized by prominent local tissue damage, including muscle necrosis. A frequent outcome of such local pathology is deficient skeletal muscle regeneration, which causes muscle dysfunction, muscle loss and fibrosis, thus provoking permanent sequelae that greatly affect the quality of life of patients. The causes of such poor regenerative outcome of skeletal muscle after viperid snakebites are not fully understood. Methodology/Principal Findings: A murine model of muscle necrosis and regeneration was adapted to study the effects of the venom and isolated toxins of Bothrops asper, the medically most important snake in Central America. Gastrocnemius muscle was injected with either B. asper venom, a myotoxic phospholipase A2 (Mtx), a hemorrhagic metalloproteinase (SVMP), or saline solution. At various time intervals, during one month, tissue samples were collected and analyzed by histology, and by immunocytochemical and immunohistochemical techniques aimed at detecting muscle fibers, collagen, endothelial cells, myoblasts, myotubes, macrophages, TUNEL-positive nuclei, and axons. A successful regenerative response was observed in muscle injected with Mtx, which induces myonecrosis but does not affect the microvasculature. In contrast, poor regeneration, with fibrosis and atrophic fibers, occurred when muscle was injected with venom or SVMP, both of which provoke necrosis, microvascular damage leading to hemorrhage, and poor axonal regeneration. Conclusions/Significance: The deficient skeletal muscle regeneration after injection of B. asper venom is likely to depend on the widespread damage to the microvasculature, which affects the removal of necrotic debris by phagocytes, and the provision of nutrients and oxygen required for regeneration. In addition, deficient axonal regeneration is likely to contribute to the poor regenerative outcome in this model. - Funding: This study was supported by NeTropica (grant 2-N-2008), by Vicerrectora de Investigacio n, Universidad de Costa Rica (project 741-A7-604). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Snakebite envenomings constitute a highly relevant and neglected public health problem on a world wide basis, affecting particularly the rural settings of Latin America, Africa and Asia [13]. In Latin America, the majority of snakebites are inflicted by species classified in the genus Bothrops [4,5]. These envenomings are characterized by complex pathological and pathophysiological profiles that include prominent local tissue damage, i.e. necrosis, hemorrhage, blistering and edema, and systemic alterations, i.e. bleeding, coagulopathy, cardiovascular shock and renal failure [610]. Among these effects, local tissue damage leading to necrosis is particularly relevant, since it is frequently followed by poor tissue regeneration, with the occurrence of permanent sequelae associated with tissue loss and dysfunction, and their consequent social and psychological implications [1,11,12]. Skeletal muscle regeneration is a complex and finely orchestrated process that involves the interaction of myogenic cells, other resident cells, inflammatory cells, blood vessels, nerves and extracellular matrix [13]. It starts after the injury of muscle fibers, with the activation of a population of quiescent myogenic cells, satellite cells, located at the periphery of the fibers between muscle sarcolemma and the basement membrane [1315]. Once activated, satellite cells become myoblasts which, after various cycles of replication, undergo a process of cell fusion leading to the formation of multinucleated myotubes which then mature to become adult myofibers [16]. For this process to proceed successfully, several requirements must be met in the microenvironment where regeneration takes place. After muscle fiber necrosis, an inflammatory reaction ensues, with invasion of the tissue by neutrophils and macrophages. Besides removing necrotic debris, these cells, especially macrophages, synthesize various cytokines and growth factors which modulate the tissue regenerative response, and also support myoblast survival in various ways [17]. Furthermore, an intact microvascular supply is required for regeneration [13], together with a restitution of the neuromuscular junction in the regenerative fibers [18]. The persistence of a structurally intact basement membrane around necrotic muscles is also important for the demarcation of the space where myoblast replication and fusion occurs [13,19]. Various muscle pathologies are associated with a good regenerative outcome, especially in conditions where blood supply and innervation are not affected, whereas in other diseases, such as ischemic lesions and some muscular dystrophies, regeneration is deficient [13,20]. Unveiling the factors associated with deficient muscle regeneration in various diseases, including snakebite envenoming, is highly relevant for the design of interventions aimed at improving this process. In the case of snake venom-induced myonecrosis, two clearly different patterns of regeneration have been described: when tissue is affected by venoms or toxins that induce muscle necrosis but do not affect the integrity of blood vessels, such as after injection of isolated myotoxic phospholipases A2 (PLA2) or cardiotoxins, regeneration proceeds successfully [2123]. Some of these myotoxins have been used as models to investigate particular aspects of muscle regeneration [21]. In contrast, when muscle is affected by venoms which, in addition to affecting muscle fibers also damage the microvasculature, inducing hemorrhage, such as many viperid snake venoms, the regenerative process is impaired, with substitution of muscle tissue by fibrosis in some areas, and with the presence of regenerating fibers of reduced diameter [22,2426]. In the case of Bothrops sp venoms, these experimental observations constitute an excellent correlate of the clinical situation, where tissue loss and dysfunction are common consequences of these envenomings [6,7,9]. From the medical standpoint (...truncated)